Conformational changes of alpha-synuclein, ABC and ECF transporters observed by high pressure EPR and DEER

Bitte benutzen Sie diese Kennung, um auf die Ressource zu verweisen: https://repositorium.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-2018020916618
Titel: Conformational changes of alpha-synuclein, ABC and ECF transporters observed by high pressure EPR and DEER
Autor(en): Sippach, Michael
Erstgutachter: Prof. Dr. Heinz-Jürgen Steinhoff
Zweitgutachter: Prof. Dr. Thomas Eitinger
Zusammenfassung: In this work two overall subjects were addressed. 1. In recent years high pressure perturbance has become a tool to investigate the folding energy landscape, the volumetric properties and the conformational equilibria of proteins. Conformational states which are not populated at ambient conditions thus become accessible to spectroscopic characterization. In this work a high pressure application was combined with EPR spectroscopy to investigate three spin labeled proteins, BSA from Bos taurus, HisJ from Salmonella enterica serovar Typhimurium and α-synuclein from Homo sapiens. The goal of these studies was to comprehend the influence of pressure on the respective EPR spectra and to identify changes in conformational equilibria and volumetric properties of the investigated proteins. Studies on BSA revealed a negative activation volume for rotational diffusion of the spin labeled site. Moreover, a rotameric equilibrium was derived from the pressure-dependent side chain dynamics and a correlating negative partial molar volume was observed, indicating a shift of the rotameric equilibrium to lesser order. In this regard it was also shown that a chaotropic medium (guanidine hydrochloride) supports the pressure-dependent effect. Spin labeled sites in the substrate binding protein HisJ revealed to be highly influenceable by low pressures between ambient conditions and 200 bar. Pressurization induced oligomerization and precipitation of the protein. Substrate binding revealed differences in pressure-dependence with regard to a decreased precipitation effect but not in relation to oligomerization. The natively unfolded protein α-synuclein plays a key role in Parkinson´s disease and is known for forming β-sheet rich aggregates, so called amyloid fibrils. The experimental data of this work revealed that hydrostatic pressure can induce a non-amyloid aggregation of monomeric α-synuclein which produces an unspecific oligomer. Furthermore, it was shown that α-synuclein amyloid fibrils can be dissolved by hydrostatic pressure. From the pressure dependent conformational equilibrium between the monomer and the fibril form the change of the partial molar volume of the investigated site was determined. 2. The second subject of this work was focused on different import systems, ATP-binding cassette (ABC) transporters and Energy-Coupling-Factor (ECF) transporters, for amino acids, vitamins and metal ions in prokaryotes. Studies on one bacterial ABC and two ECF transporter systems from two different organisms, the histidine ABC-type transporter HisQMP2 from Salmonella enterica serovar Typhimurium, the biotin ECF-type importer BioMNY from Rhodobacter capsulatus and the cobalt-specific ECF-type transporter CbiMNQO from Rhodobacter capsulatus, were performed using DEER and cw EPR spectroscopy. The goal of the studies on HisQMP2 and BioMNY was to shed light on conformations and dynamics connected to their transporter function. Studies on CbiMNQO aimed at the detection of the substrate in the transporter´s substrate binding unit. For HisQMP2 transport cycle dependent conformational changes and interactions with the substrate binding protein HisJ were revealed. Three different distance values between sites H101R1 and H101’R1 in the transporter´s nucleotide binding domains were assigned to the apo-, the ATP-bound and the posthydrolysis state. It was shown that the closed conformation of the nucleotide binding domains is achieved only in the presence of the ligand-bound HisJ which indicates a transmembrane communication of the association of HisJ to the transporter. Furthermore, interspin distances were determined between sites D86R1-A96R1, C197R1-C104R1 and A118R1-G123R1 in the transmembrane domains HisQ and HisM revealing distinguishable conformational states which correlate to the different states of the nucleotide binding sites during the hydrolysis cycle. Measured interspin distances between HisJ and HisM in the HisQMP2 complex showed that interaction only occurred in the closed state of the HisP2 dimer, the nucleotide bound state. Two different, substrate-dependent interactions between site G24R1 in HisJ and site A96R1 in HisQMP2 were observed, revealing that the substrate-free and substrate-bound form of HisJ both associate with HisQMP2. Distance measurements between sites G24R1 and T151R1 in HisJ in the presence and absence of its substrate revealed interspin distance changes that correlate with the proteins open and closed conformation. Investigations on the ECF transporter BioMNY, reconstituted into nanodiscs, revealed a closure and reopening of the nucleotide binding domains between sites H87R1 and H87’R1 using DEER spectroscopy which delivered interspin distance values that correlate with the apo-, the ATP-bound and the posthydrolysis state of the transporter. Further experiments were aimed to shed light on the transporters substrate-translocation mechanism with regard to the so called toppling over mechanism. Unfortunately, the experiments of this work were not able to give a distinct answer with respect to the proposed model because of the transmembrane domains tendency to oligomerize when reconstituted into nanodiscs. In this work we showed that substrate uptake by the substrate binding unit CbiM of the cobalt-specific ECF transporter CbiMNQO depends on the presence of the small transmembrane protein CbiN. Measurements of spin labeled CbiMN in detergent showed oligomerization of CbiM.
URL: https://repositorium.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-2018020916618
Schlagworte: Electron paramagnetic resonance spectroscopy; High Pressure; ABC transporter; ECF transporter; Alpha-synuclein
Erscheinungsdatum: 9-Feb-2018
Enthalten in den Sammlungen:FB04 - E-Dissertationen

Dateien zu dieser Ressource:
Datei Beschreibung GrößeFormat 
thesis_sippach.pdfPräsentationsformat7,09 MBAdobe PDFMiniaturbild
Öffnen/Anzeigen


Diese Ressource wurde unter folgender Copyright-Bestimmung veröffentlicht: Lizenz von Creative Commons Creative Commons